In this continuation request we intend to focus exclusively on one of only two recognized thyroid-specific gene associations, namely the TSH receptor gene, which codes for the predominant antigen for Graves' disease. In Specific Aim 1 we will extend our data on TSR intron 1 SNPs associated with Graves' disease. We hypothesize that less common TSH receptor SNPs will be found in intron I of the gene which specifically associate with Graves' disease and confer a higher relative risk than currently recognized SNPs. To achieve this, we will proceed to deep re-sequencing of the TSHR gene to discover these less common SNPs. In order to find increased risk associations we will also examine TSHR gene interactions with a number of additional, selected, AITD susceptibility genes. We will examine gene interactions between the TSHR and other susceptibility genes and epigenetic phenomena including the CTLA-4 and IL-23 receptor genes, the HLA DR-3 haplotypes and X chromosome inactivation (XCI). In Specific Aim 2 we will determine changes in TSHR mRNA splicing determined by associated SNP polymorphisms. We have previously reported on the extensive degree of TSHR mRNA splicing in the human thyroid with two major mRNA variants of 1.3 and 1.7 Kb. Here we will determine the entire TSHR mRNA splicing repertoire and the transcription of these variants in relation to associated TSHR SNPs such as found in intron 1. In Specific Aim 3 we will examine the role of small RNAs encoded by TSHR intron 1. Another major way by which intronic regions may influence gene action is by the endogenous production of small non-coding microRNAs (miRNAs) which may regulate gene expression. We recently used a computational approach to predict potential regions of the human TSHR-intron 1 that may harbor miRNAs and detected 3 candidates variably expressed in small RNA preparations from a variety of tissues, including human thyroid, and confirmed them by DNA sequencing. There are intronic polymorphisms within the pre-miRNAs of these candidates which may influence the biogenesis of intron 1 miRNAs and their ultimate function. Small RNAs may influence TSHR function by transcriptional regulation resulting in stimulation or depression of TSHR expression and/or function. We will examine this possibility directly by TSHR gene targeting and indirectly by using thyroid cell transient siRNA transfection models with TSHR expression and signal transduction end points in human and rat thyroid cell lines. PUBLIC HEALTH RELEVANCE: The potential benefits of this study are the identification of TSHR gene markers for Graves' Disease and the improved diagnosis and classification of the disorder. The benefit to the individual and society is potentially great in alleviating suffering due to the more severe forms of this thyroid disorder, especially Graves' ophthalmopathy.